This invention relates to the measurement of blood coagulation factor XIII in plasma. More particularly, the invention relates to a process for measuring the plasma factor XIII by determining the activity of XIIIa, an activated form of the factor XIII, by a fluorescence method in which reacted fluorescent material and non-reacted fluorescent material are separated by means of molecular sieve chromatography to accurately measure the amount of the reacted fluorescent material.
Plasma factor XIII is an enzyme which is effective in the final stage of blood coagulation. More precisely, it is categorized as a non-active enzyme which shows its enzymatic activity only when it is converted into form XIIIa by the action of thrombin and Ca.sup.++ in plasma. It is therefore possible to determine the amount of plasma factor XIII by measuring the activity of XIIIa.
The action of XIIIa in the blood coagulation mechanism consists in forming intermolecular crosslinkage, in other words, isopeptide bonds between fibrins produced by the action of thrombin, and thus XIIIa is deemed to have a transglutaminase activity. Because of these peculiarities, this plasma factor has a very great clinical significance, and its importance is acknowledged especially in the detection of disseminated intravascular coagulation (DIC) and kidney diseases and in the clinical examination in the fields of surgery and obstetrics. Incidentally, a report by the DIC research group of the Ministry of Public Welfare backs up such utility of plasma factor XIII. The development of a simple and accurate method of measuring this plasma factor XIII, therefore, has been strongly desired.
Various methods such as mentioned below have been deviced for the measurement of plasma factor XIII:
(1) Clot test method: The solubility of fibrin gel in urea or an acid is examined. This method is simple but rather lacks accuracy of determination.
(2) Crosslink determination method: Crosslinked lysine in fibrin gel is determined. This method requires a large volume of specimen and is also unsatisfactory in quantitative accuracy.
(3) Radioisotope method: The amount of a .sup.14 C compound incorporated into casein is measured. This method is excellent in determination accuracy and high in sensitivity but has drawbacks mentioned below.
(4) Fluorescence method: Incorporation of dansylcadaverine into casein is determined. This method is high in both measurement accuracy and sensitivity but rather complicated in operation.
(5) Antigen-antibody method: This method is of a medium degree in both sensitivity and accuracy of determination but has a problem that the antigen activity and the enzyme activity are not always consistent with each other because of a wide difference in antigenecity between the factor XIII and its activated form XIIIa.
Thus, a variety of methods have been proposed for measurement of the factor XIII, among which the radioisotope (RI) method and fluorescence method are believed to be excellent in both sensitivity and accuracy of determination. However, the RI method necessitates a substantial investment for the equipment as a radioactive substance is treated. This method is also complicated in operation and unsuited for a small-scale practice. The fluorescence method is free of such problems of the RI method but still has some serious disadvantages as discussed below. This method was first reported by Lorand et al in 1969 (J. Clin. Invest., 48: 1054-1064, 1969). According to this method, a casein-dansylcadaverine complex is formed by bonding .gamma.-glutamyl groups of casein and .epsilon.-amino groups of dansylcadaverine by the action of XIIIa as depicted by the following formula: ##STR1##
This casein-dansylcadaverine complex is precipitated by using a strong acid and repeatedly washed with an organic solvent. After separating unreacted dansylcadaverine by washing, the precipitate is dissolved in an aqueous solution of urea containing a surfactant and the fluorescence intensity of the solution is measured. From the measured value of fluorescence intensity, both the activity of XIIIa and the quantity of the factor XIII are determined.
This method, although excellent in many points, is yet unsatisfactory in some respects. It is an essential requirement in this method to separate the caseindansylcadaverine complex free of unreacted dansylcadaverine, which necessitates use of a strong acid and an organic solvent. Use of such matters, however, is undesirable in a practical test. Also, washing of the precipitate with an organic solvent is troublesome, and much time and labor is often required for the perfect separation of entrapped unreacted dansylcadaverine. Because of these defects, this method could hardly be applied to the routine clinical tests.